Dr. Pastorino has extensive experience and publications in research on cell injury. The candidates goal is to become an independent investigator in the mechanisms by which ethanol brings about cell injury. Dr. Pastorino is a member of the Alcohol Research Center at Thomas Jefferson University. This environment offers him numerous opportunities to interact with established investigators in alcohol research. The research career development plan involves 6 components 1). Planning of experiments and interpretation of data, 2). Acquisition and training of laboratory personnel, 3). Grant management. 4). Handling of hazardous and radioactive materials, 5). Humane treatment of laboratory animals 6.) preparations of manuscripts and grant applications. Chronic ethanol exposure promotes the development of alcoholic liver disease (ALD) but the mechanisms underlying ethanol- induced hepatotoxicity remain poorly understood. Proinflammatory cytokines such as tumor necrosis factor alpha (TNFalpha) have been linked to many of the associated damage and repair processes seen in ALD. TNFalpha prompts the opening of the mitochondrial permeability transition pore (MPT). The MPT is the opening of a large, nonspecific pore across the outer and inner mitochondrial membrane. In preliminary results we demonstrate that a 48h exposure of HepG2 cells to ethanol, enhanced susceptibility to TNFalpha cytotoxicity. A similar enhancement of TNFalpha cytotoxicity is observed in primary cultured hepatocytes isolated from chronically ethanol-fed rats. The data suggest that ethanol enhances TNFalpha cytotoxicity, at least in part, by promoting the MPT. The general goals of this project are to investigate the mechanisms by which ethanol potentiates TNFalpha induced cytotoxicity. Our working hypothesis is that ethanol inhibits signaling pathways that protect against TNFalpha cytotoxicity and activates pathways which cause the mitochondrial permeability transition. This theory is supported by our preliminary observations that ethanol inhibits P13kinase dependent BAD phosphorylation induced by TNFalpha and promotes cell death in a p38 MAPK dependent and caspase 8 independent manner. Our SPECIFIC AIMS are to #1) define the role of ethanol in the alteration of PI3-kinase activity and its influence on TNFalpha cytotoxicity, #2) delineate the role of p38 MAPK activity in ethanol and TNFalpha cytotoxicity; and #3) delineate the mechanism by which TNFalpha plus ethanol activate Group II caspases.